EP0302429B1 - Menschliches Interleukin-4, Expressionsvektoren dafür und diese enthaltende Transformanten - Google Patents

Menschliches Interleukin-4, Expressionsvektoren dafür und diese enthaltende Transformanten Download PDF

Info

Publication number
EP0302429B1
EP0302429B1 EP88112458A EP88112458A EP0302429B1 EP 0302429 B1 EP0302429 B1 EP 0302429B1 EP 88112458 A EP88112458 A EP 88112458A EP 88112458 A EP88112458 A EP 88112458A EP 0302429 B1 EP0302429 B1 EP 0302429B1
Authority
EP
European Patent Office
Prior art keywords
human
cells
human interleukin
dna
molecular weight
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP88112458A
Other languages
English (en)
French (fr)
Other versions
EP0302429A1 (de
Inventor
Tasuku Honjo
Kazuaki Hama
Akiyoshi Kawasaki
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ono Pharmaceutical Co Ltd
Original Assignee
Ono Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ono Pharmaceutical Co Ltd filed Critical Ono Pharmaceutical Co Ltd
Priority to AT88112458T priority Critical patent/ATE92110T1/de
Publication of EP0302429A1 publication Critical patent/EP0302429A1/de
Application granted granted Critical
Publication of EP0302429B1 publication Critical patent/EP0302429B1/de
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5406—IL-4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F02—COMBUSTION ENGINES; HOT-GAS OR COMBUSTION-PRODUCT ENGINE PLANTS
    • F02B—INTERNAL-COMBUSTION PISTON ENGINES; COMBUSTION ENGINES IN GENERAL
    • F02B75/00—Other engines
    • F02B75/02—Engines characterised by their cycles, e.g. six-stroke
    • F02B2075/022—Engines characterised by their cycles, e.g. six-stroke having less than six strokes per cycle
    • F02B2075/027—Engines characterised by their cycles, e.g. six-stroke having less than six strokes per cycle four

Definitions

  • the present invention relates to novel human IL4 having immune-activating effects, novel expression vectors for said human IL4 and novel transformants thereof.
  • Interleukin 4 is a glycoprotein produced by T lymphocytes when stimulated with lectin, phorbol ester or antigen.
  • IL4 has various significant effects on the immune response, such as differentiation and proliferation, of B lymphocytes, of T lymphocytes and of mast cells (see Y. Noma et al., Nature, 319, 640-646, 1986 (hereafter “Reference A”); F. Lee et al., Proc. Natl. Acad. Sci. USA, 83, 2061-2065, 1986 (hereafter “Reference B”); E. Severinson et al., Eur. J. lmmunol., 17, 67-72, 1987 and T. R.
  • IL4 has such strong immune-activating effects, it is believed that IL4 is useful for the prevention and treatment of infections, AIDS, cancer, immune deficiency, etc. (see R. Fernandez-Botran et al., Proc. Natl. Acad. Sci. USA, 83, 9689-9693, 1986 and R. Palacios et al., EMBO J., 6, 91-95, 1987).
  • Native human IL4 can be obtained by treating normal human peripheral blood lymphocytes, malignant human T cell lines or human T cell hybridoma with drugs.
  • physical and chemical characteristics e.g. the total molecular weight, molecular weight of the sugar chain, the isoelectoric point, etc.
  • IL4 is expressed in monkey cells transiently, and the cells die after producing human IL4.
  • the cells used are not available for expression again, and it is necessary to always transfect fresh cells.
  • a number of DNA vector and a good deal of work are required for transfection.
  • the method using monkey cells is not suitable for supplying enough human IL4 for clinical use.
  • an object of the present invention is to provide large quantities of human IL4 useful for the prevention and treatment of infections, AIDS, cancer, immunodeficiency, etc.
  • Another object of the present invention is to provide an expression vector for the production of large quantities of human IL4.
  • Still another object of the present invention is to provide transformants which express large quantities of human IL4.
  • a novel human IL4 having specific physical and chemical characteristics produced by utilizing an expression vector which comprises a DNA sequence encoding human IL4 at a position downstream from an SV40 early promoter, and a DNA sequence encoding dihidrofolate reductase (hereafter "DHFR”) at a position downstream from the promoter, and by utilizing dihydrofolate reductase-defective Chinese hamster ovary cells (hereafter "CHO cells”), as host cells.
  • DHFR dihidrofolate reductase
  • an expression system with high efficiency has been found in the present invention comprising the combination of an expression vector containing SV40 early promoter and CHO cells. It has also been found in the present invention that by incorporating the gene of DHFR in the expression vector and further by using DHFR-defective cells, an expression system with more high efficiency can be obtained.
  • novel human IL4 produced by the expression system of the present invention has specific physical and chemical characteristics (e.g. molecular weight of the sugar chain, i.e. the sugar content of the human IL4, etc.)
  • each protein has quite different sugar chain, length of the chain, sugar components, etc. These differences are represented by the molecular weight of the sugar chain, etc, as shown in Table I below.
  • glycoproteins having sugar components with the same characteristics are produced, when the expression system using CHO cells is applied to the expression of various glycoproteins.
  • the present invention provides in particular:
  • the human IL4 of the present invention contains 129 amino acids from the 25th amino acid, histidine to the 153rd amino acid, serine, and lacks amino acids from the first amino acid, methionine to the 24th amino acid, glycine (see Examples 8 and 9 described hereinafter).
  • the sugar content of the human IL4 of the present invention is quite distinct from that of the human IL4 known to date.
  • the molecular weight of the human IL4 of the present invention is 17 to 19 kd (kilodaltons).
  • the molecular weight of only the protein moiety is 14962. Therefore, the difference in molecular weight between total human IL4 and the protein moiety, i.e. 2000 to 4000, preferably 2500 to 3500, is considered to be the molecular weight of the sugar content.
  • the molecular weight of human IL4 expressed by monkey cells is 22 kd, and hence that of the sugar content is considered to be about 7 kd. Therefore, it is confirmed that the quantity of the sugar content bonded to the protein moiety in the human IL4 expressed by monkey cells is more than two times that in the human IL4 of the present invention. In addition, there is a great difference in molecular weight between the sugar content of both human IL4s.
  • the molecular weight of human IL4 expressed by yeast is 60 kd and that of the sugar content is considered to be 45 kd.
  • the molecular weight of the sugar content of the human IL4 expressed by yeast is greatly different from that of the present invention.
  • the human IL4 of the present invention preferably has a isoelectric point of 8.5 to 10.5, more preferably 9.8 to 10.1.
  • the human IL4 of the present invention has a sugar content which comprises at least mannose, fucose and galactose as neutral sugars, at least glucosamine as an amino sugar and at least N-acetylneuraminic acid as a sialic acid.
  • the human IL4 of the present invention preferably has a sugar content wherein mannose, fucose, galactose, glucosamine and N-acetylneuraminic acid are contained in amounts of 3.0 to 4.0, 1.0 to 2.0, 2.5 to 3.5, 4.5 to 5.5 and 1.5 to 2.5, mols per total mol of sugar, respectively.
  • the human IL4 of the present invention is sufficiently pure, but is not necessarily a single chemical compound.
  • the present invention may comprise many homogenous human IL4 having various characteristics within the scope described above and heterogeneous human IL4 having slightly different sugar contents.
  • the present invention also includes the expression vector for human IL4 and the transformant.
  • the expression vector for human IL4, of the present invention may be prepared by ligating:
  • the DNA of human IL4 may be prepared by (1) extracting mRNA from cells being capable of producing IL4, such as normal human lymphycytes, (2) constructing cDNA by using reverse transcriptase, and, then, (3) cloning the cDNA from the obtained cDNA library in Escherichia coli.
  • the desired cDNA may be selected from a cDNA library by cross-hybridization using a part of the cDNA of mouse IL4 (described in Reference A and B) as a probe or by using the biological activities of human IL4 as a screen. Efficient selection is a obtained by using both of the above methods together. It is also possible to select by hybridization using a synthetic oligonucleotide having a partial nucleotide sequence of cDNA of human IL4, which has already reported (described in Reference C), as probe.
  • nucleotide sequence rich in adenine (A) and uracil (U) exists in a non-translated region at the 3'-side of mRNA of lymphokines such as IL4 and that this sequence relates to the instability of mRNA itself (see G. Shaw et al., Cell, 46, 659-667, 1986). It is considered by the present inventors that in IL4, three groups of the nucleotide sequence shown by "ATTTA" (i.e., from 472nd to 476th, from 506th to 510th and from 514th to 518th), correspond to that relating to instability.
  • ATTTA three groups of the nucleotide sequence shown by "ATTTA" (i.e., from 472nd to 476th, from 506th to 510th and from 514th to 518th)
  • the DNA of human IL4 wherein the nucleotide sequence for instability is removed may be prepared by (1) cleaving the vector DNA containing the cDNA of human IL4 by a restriction enzyme which recognizes a particular nucleotide sequence existing singly at a position downstream from the nucleotide sequence for instability, e.g. Sal I, (2) digesting with a nuclease such as Bal 31, and ,then, (3) cloning the obtained DNA fragment into Escherichia coli.
  • a restriction enzyme which recognizes a particular nucleotide sequence existing singly at a position downstream from the nucleotide sequence for instability
  • the purpose of the present invention may be accomplished.
  • the expression "the DNA where at least the nucleotide sequence for instability is removed” means that three groups of nucleotide sequences shown by "ATTTA" in the 3' non-translated region of the DNA are removed alone or along with some or all of the remaining 3' non-translated region. However, it is preferable to use the DNA wherein at least the nucleotide sequence for instability is removed.
  • any vectors containing an SV40 early promoter may be used.
  • examples of such vecters are pSV2 vector (see R. C. Mulligan et al., Science, 209, 1422-1427, 1980) and pKCR ⁇ H 2 vector (see K. O'Hare et al., Proc. Natl. Acad. Sci., USA, 78, 1527-1531, 1981 ).
  • pKCR• H 2 vector is preferred.
  • pKCR• H 2 vector has a splicing sequence and polyadenylation sequence, both derived from the gene of ,8-globin, besides an SV40 early promoter. Therefore, it is suitable for the expression of the desired product in mammalian cells. Furthermore, this vector has an origin of replication in Escherichia coli and an ampicillin resistance gene as a selection marker, in order to enable the replication in Escherichia coli. Hence, pure vector DNA may be prepared in quantity by using Escherichia Coli.
  • the DHFR gene may be prepared by the method known (see R. J. Kaufman et al., Molecular and Cellular Biology, 2, 1304-1319, 1982).
  • promoters for the expression of the DHFR gene any promoters operating in eukaryotic cells, such as an SV40 early promoter, metallothionein gene promoter, adenovirus gene promoter, retrovirus gene promoter etc. may be used.
  • the expression vector for human IL4 may be prepared, and the preparation may be carried out by conventional methods well-known to those skilled in the art (see T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982).
  • CHO dhfr- cells Dihydrofolate reductase-defective Chinese hamster ovary cells (hereafter "CHO dhfr- cells") for the transfection of the expression vector for human IL4 are described in G. Urlaub et al., Proc. Natl. Acad. Sci. USA, 77, 4216-4220, 1980.
  • the transfection of the vector DNA into CHO dhfr- cells may be carried out by methods known, for example, by the calcium phosphate method (described in F. L. Graham et al., Virology, 54, 536-539, 1973).
  • the above cells of the present invention in which the expression vector was transfected, may be selected by utilizing the property that the transformant can grow even in a culture medium for selection in which the parent CHO dhfr- cells die for lack of thymidine and hypoxanthine.
  • the transformant in which the DHFR gene is amplified by the action of an antagonistic inhibitory agent for DHFR may be easily distinguished from that in which DHFR gene is not amplified, because the former can grow in a culture medium containing an antagonistic inhibitory agent for DHFR, e.g. methotrexate.
  • One of the transformants of the present invention obtained in this way which was named CHO-B91 cells, has been deposited at the Institute for Fermentation, Osaka, Japan (IFO) with deposit No. IFO 50137 and deposited at the Fermentation Research Institute, Ibaraki, Japan (FRI) with deposit No. BP-1752.
  • the transformant of the present invention which produces human IL4 efficiently and in quantity, may be cultured in a conventional culture medium (for example, a-MEM containing 10%(v/v) fetal bovine serum) in the presence or absence of methotrexate.
  • a conventional culture medium for example, a-MEM containing 10%(v/v) fetal bovine serum
  • the desired protein, secreted into the culture medium may be purified by conventional methods, for example salting-out techniques, ion-exchange chromatograpy, gel- filtration methods, hydrophobic column chromatograpy, affinity chromatograpy, etc.
  • the human IL4 obtained by the present invention improves the immune response and hence is useful for the treatment and prevention of diseases which require such improvement, for example cancer, infections, AIDS, functional immune deficiency, etc.
  • the suitable dose may vary with the conditions of disease, age, sex and body weight, of the subject.
  • a suitable dosage is between 0.1 ⁇ g to 1 mg for intravenous administration.
  • the stimulatory activity on human lymphocyte growth was used as an indication of immune-activating effects of human IL4 in the present invention.
  • the stimulatory activity on human lymphocyte growth was measured by the following method.
  • Lymphocytes prepared from human peripheral blood were suspended in the culture medium (RPMI-1640 containing 10%(v/v) fetal bovine serum and 5 x 10- 5 M 2-mercaptoethanol) containing phytohemagglutinin (10 ⁇ g/ml), and cultured for 4 to 6 days at 37°C in the presence of 5% C0 2 .
  • the cultured lymphocytes were washed with the culture medium (more than three times), and 5 x 10 4 lymphocytes thus obtained were again suspended in 200 ⁇ l of the culture medium.
  • the suspension was further cultured for three days. It was pulsed with tritium labeled thymidine (0.25 ⁇ Ci/culture) for 12 hours before termination of culturing. Then, the incorporation of thymidine into the high molecular fraction were measured by a liquid scintillation counter. The relative activity measured was 2.5 - 10 x 10 5 U/mg of protein.
  • antibody against human IL4 may be prepared by using human IL4 of the present invention as an antigen, and receptors of human IL4 can be isolated and purified by using the human IL4 as a ligand.
  • Human peripheral blood lymphocytes were suspended in RPMI 1640 medium supplemented with 10%-(v/v) fetal bovine serum (hereafter "FBS”), 5 ⁇ 10 -5 M of 2-mercaptoethanol and 1 ⁇ g/ml of phorbol-12- mylistate-13-acetate (hereafter "PMA”), and then incubated at 37 °C in 5% C0 2 for 12 hrs.
  • FBS fetal bovine serum
  • PMA phorbol-12- mylistate-13-acetate
  • human peripheral blood lymphocytes were cultured in RPMI-1640 supplemented with 10%(v/v) FBS, 5 ⁇ 10 -5 M of 2-mercaptoethanol and 10 ⁇ g/ml of phytohemagglutinin, at 37 ° C in 5% C0 2 for four to six days. These lymphocytes were washed (over three times) with medium and 5 ⁇ 10 4 cells were resuspended into 200 ⁇ l of medium which contained the sample to be examined. The incubation was continued for additional three days.
  • the cells were pulse-labeled with [ 3 H]-thymidine (0.25 ⁇ Ci/culture) to measure the incorporation of the radioactivity into a high molecular weight fraction of the cells.
  • human lymphocytes prepared from human tonsils were purified using the rosetta formation using 2-aminoethylisothiouronium bromide treated sheep red blood cells (SRBC) to remove T lymphocytes.
  • SRBC 2-aminoethylisothiouronium bromide treated sheep red blood cells
  • the purified B-lymphocytes obtained were incubated in RPMI-1640 supplemented with 10%(v/v) FBS, 5 ⁇ 10 -5 M of 2-mercaptoethanol and 5 ⁇ g/ml of anti-IgM antibody bound glass beads, at 37 °C in 5% C0 2 for two to three days.
  • Anti-IgM antibody bound beads were removed by density gradient centrifugation using lymphoprep (trade name, product of Nyegaard Inc.).
  • B-lymphocytes were washed with the medium and then 5 ⁇ 10 4 cells were resuspended in 200 ⁇ l of medium each. Sample supernatants were added into this cell suspension and the mixture was allowed to incubate for the additional two days. The activity was measued by [ 3 H]-thymidine incorporation as described above.
  • the cloning plasmid pCDV 1 -PL (described in Reference A) was digested with the restriction enzyme Kpnl. To the 3'-ends of this fragment, an oligo dT chain, average of 45 bases, was added with terminal deoxynucleotidyl transferase. This fragment was digested with EcoRl and the larger fragment was recovered from an agarose gel. Upon passing the larger fragment through an oligo (dA) column, the purified vector- primer was obtained.
  • Plasmid pSP62-K2 (described in Reference A) was digested with Sacl. To the 3'-ends of this fragment, an oligo dG chain, average of 14 bases, was added with terminal deoxynucleotidyl transferase. This fragment was digested with Hindlll to obtain the linker DNA as the smaller fragment.
  • a cDNA was synthesized with 2 ⁇ g of vector primer as a template with 10 units of reverse-transcriptase.
  • an oligo dC chain average of 14 bases, was added with terminal transferase.
  • the primer was digested with Hindlll, the cDNA fragment was ligated with the linker.
  • the first strand, RNA was made into DNA with E. Coli polymerase I and R Nase H.
  • E. Coli HB101 was transformed with the plasmid thus obtained to prepare a cDNA library which contained about one million independent clones.
  • Plasmid pSP6 KmIL4-374 (described in Reference A), which carried mouse IL4 cDNA, was digested with BamHl to excise an about 800 base pair insert. This fragment was further digested with Bal 31 in 40 ⁇ l of reaction mixture which contained 35 ⁇ g of DNA, 12 mM of CaCI 2 , 12 mM of MgCl 2 , 0.2 M of NaCI, 20 mM of Tris-HCI buffer (pH 8.0), 1 mM of EDTA and 0.3 units of Bal 31, at 30 °C for 3 min. After the mixture was extracted the phenol, DNA was precipitated with ethanol, and then a BamHl linker was ligated with T4- ligase.
  • the BamHl treated DNA fragment was inserted in the BamHl site of pUC 18 to prepare a plasmid pSP6mIL4 ⁇ .
  • the 5'-fragment obtained with BamHl/Rsal digestion does not contain any part of SP6 vector.
  • the Rsal/Rsal fragment was prepared, which covered most of the protein coding region.
  • the cDNA library described above was adhered to GSN filters (product of German Science Inc.) to prepare four replica filters. Two fragments prepared from mouse IL4 cDNA, BamHl/Rsal (120 bp) and Rsal/Rsal (373 bp) were nick-translated independently to yield highly labeled probes (about 5 x 10 8 cpm/ ⁇ g).
  • Two replica filters were hybridized with each probe independently (50mM Tris-HCI buffer (pH 7.8), 10 mM EDTA, 1 M NaCI, 0.1%(w/v) BSA, 0.1%(w/v) polyvinylpyrrolidone, 0.1%(w/v) Ficol 400, 0.1%(w/v) SDS and 50 ⁇ g/ml of sonicated salmon sperm DNA) at 37 °C for 24hrs.
  • Plasmid pSP(6-127) was digested with BamHl to excise a 0.8 Kb fragment which contained the whole sequence of human IL4. After treatment with Klenow fragment and Hindlll linker attachment, the DNA was inserted into Hindlll site of pKCRH 2 (described in K. O'Hare et al., Proc. Natl. Acad. Sci. USA, 78, 1527-1531, 1981) to prepare plasmid pKCRhIL4 (see Figure 2).
  • Plasmid pSV2neo (described in R. C. Mulligan et al., Science, 209, 1422-1427, 1980) which can express the neomycin resistance gene neo in mammalian cells, was digested with Accl and EcoRl to prepare a 2.5 Kb fragment, which carried neo gene. The excised neo gene was treated with Klenow fragment and a BamHl linker attached. The DNA was then inserted in the BamHl site of plasmid pMDO which can express dihydrofolate reductase (described in S. K. Kim et al., Cell, 42, 129-138, 1985) to prepare plasmid pDneo (see Figure 2).
  • DHFR and neo are arranged in the same direction.
  • pDneo was digested with EcoRl, treated with Klenow fragment and then a Sall linker attached .
  • Sall a 6.5 Kb fragment purified was inserted into the Sall site of plasmid pKCRhlL4 to obtain the expression vector pKCRhIL4-Dn (see Figure 2).
  • Plasmid pSP(6-127) was digested with Sall, and then further digested with 0.26 unites of Bal 31 (3.5 ⁇ g DNA/40 ⁇ l reaction mixture) at 30 ° C.
  • Bal 31 3.5 ⁇ g DNA/40 ⁇ l reaction mixture
  • Nine ⁇ l of a sample solution was mixed in 1 ⁇ l of 0.2M EGTA throughout the time course(2, 4, 6 and 10 minutes).
  • An aliquot (2 ⁇ l) was digested with EcoRl and analyzed by PAGE. DNA of one pool which gave rise to a fragment around 100 bp (4 minutes reaction) was treated with Klenow fragment, ligated with an Xhol linker, recirculized and then incorporated to DH 1 to yield about 2,000 transformants.
  • the plasmids were obtained from 120 independent clones and digested with EcoRl and Xhol to examine for the excision of the 100 bp fragment. Twenty-eight clones were selected and partially sequenced. One of them, plasmid pSP(6-127)A carried no 3'-noncoding sequence, whose TGA terminal codon and Xhol linker were directly ligated (see Figure 3). The fact shows that the entire nucleotide sequence downstream from the signal sequence for the termination of translation was removed.
  • Plasmid pSP(6-127)A was digested with BamHl and Xhol to excise a 0.5 Kb fragment. Upon treatment with Klenow fragment and attachment of a Hindlll linker, the IL4 coding region was inserted into the Hindlll site of plasmid pKCRH 2 to obtain plasmid pKCRhIL4A (see Figure 3).
  • Plasmid pMDO described in Example 2 was digested with EcoRl, treated with Klenow fragment, and a Sall linker attached. The 3.5 Kb of Sall fragment which contained DHFR gene was then inserted into the Sall site of plasmid pKCRhIL4A to obtain the plasmid pKCRhIL4A-D (see Figure 3).
  • the plasmid DNA, pKCRhIL4-Dn was linearized by Pvul digestion. About 1 x 10 6 CHO dhfr- cells (G. Urlaub et al., Proc. Natl. Acad. Sci. USA, 77, 4216-4220, 1980) in a 10 cm dish were transfected with 20 ⁇ g of the linearized DNA by the calcium phosphate co-precipitation method (F.L. Graham et al., Virology, 54, 536-539, 1973).
  • aMEM Flow laboratories
  • aMEM Flow laboratories
  • selection medium aMEM containing 10%(v/v) dialyzed FBS (GIBCO) without hypoxanthine and thymidine
  • CHO dhfr - cells were also transfected with the plasmid DNA, pKCRhIL4A-D, in the same manner and grown in selection medium for 2 weeks. A hundred colonies were isolated and the production level of human IL4 for each clones was determined. Table III shows the activities of 10 representative clones, which were relatively well grown.
  • Human IL4 production in the cells transfected with pKCRhIL4A-D was about 30 fold higher than that in the cells transfected with pKCRhIL4-Dn.
  • clones (clone No. A-8 and -10) from group A and 6 clones (clone No. B-1, -2, -3, -4, -9 and -10) from group B were selected and about 7 ⁇ 10 5 cells of each clone were placed into a 10 cm disk and cultured in the selection medium containing 50 nM of methotrexate (hereafter "MTX") for 2 to 4 weeks. After the cells were grown to confluence, the IL4 activity in the medium was assayed as described above.
  • MTX methotrexate
  • IL4 production in clones A-10 and B-1 was greatly enhanced after MTX treatment. 20 subclones were isolated from these clones.
  • Enhancement of IL4 production of clone B-9 was not observed by the treatment of 50 nM MTX.
  • the cells were treated in the presence of 200 nM MTX in the same procedure, and 10 subclones were isolated.
  • B-9-1 One of the high producer cell lines, B-9-1, was named as CHO-B91 and has been deposited at the Institute for Fermentation, Osaka, Japan (IFO) with deposit No. IFO 50137 and deposited at the Fermentation Research Institute, Ibaraki, Japan (FRI) with deposit No. BP-1752.
  • microcarrier beads cytodex 3, Pharmacia
  • PBS(-) phosphate buffered saline
  • FBS fetal bovine serum
  • the cells were grown in a monolayer on the surface of the microcarrier beads and washed with SFM101 medium to remove FBS.
  • the cells were maintained for another 24 days by feeding fresh medium and harvesting spent medium every day at 500 ml/day.
  • Example 5 Twelve liters of the culture supernatants obtained in Example 5 were cooled to 4 ° C, and acidified by adding 120 ml of 10%(v/v) trifluoroacetic acid (hereafter "TFA"). After adding thereto 120g of Sepralyte C-1 (trade name, prepared by Analytichem International Co.) pre-washed with acetonitrile, the mixture was stirred slowly for two hours. The obtained mixture was allowed to stand for 30 minutes and the supernatants were removed therefrom to recover the beads on glass filter (3G).
  • TFA 10%(v/v) trifluoroacetic acid
  • the recovered beads were washed with a total volume of two liters of 30%(v/v) acetonitrile (containing 0.1%(v/v) TFA) several times at 4 °C, and then washed similarly with the total volume of 400 ml of 50%(v/v) acetonitrile (containing 0.1% TFA(v/v)) to wash out the absorbates.
  • the obtained solution was concentrated under reduced pressure below 40 ° C to remove the acetonitrile, and was absorbed on cation-exchange column (Mono S, trade name, prepared by Pharmacia Co.) pre-equilibrated with 50 mM phosphate buffer (pH 6.0).
  • the column was washed by adding 50 mM phosphate buffer (pH 6.0) for 10 minutes at a flow rate of 2 to 3 ml/min, and then washed by adding 50 mM phosphate buffer (pH 6.0) containing 0.3M sodium chloride until the absorbance (280 ⁇ m) become below 0.1.
  • the desired substance was eluted with 50 mM phosphate buffer (pH 6.0) containing 0.4 M sodium chloride as eluent with monitoring by using a Phast System (trade name, prepared by Pharmacia Co.) to collect fractions having a molecular weight of 18 to 20 kd.
  • the collected fractions were diluted ten times with 50 mM phosphate buffer (pH 6.0) and again absorbed on cation-exchange column.
  • the column was washed by adding 50 mM phosphate buffer (pH 6.0) containing 0.3 M sodium chloride until the absorbance (280 ⁇ m) became below 0.02, and then the NaCl concentration was increased linealy from 0.3 M to 0.5 M over 30 min.
  • the fraction was also monitored with UV absorption and sodium dodecylsulfate/polyacrylamide gelelectrophoresis (hereafter "SDS/PAGE").
  • Ammonium sulfate was dissolved in the collected fractions at a final concentration of 1.7 M and the solution was absorbed on hydrophobic column chromatography (Phenyl. Superose, trade name, prepared by Pharmacia Co.) pre-equilibrated with 50 mM phosphate buffer (pH 6.0) containing 1.7 M ammonium sulfate, and then the concentration of ammonium sulfate was decreased linearly from 1.7 M to 0.0 M over 60 min. with monitoring by UV absorption and SDS/PAGE to collect fractions having a molecular weight of 18 to 20 kd. Mainly broad fractions at 0.5 to 0.3 M were obtained.
  • the fraction having a single band was collected and concentrated by using ultrafiltration (YM-10, trade name, prepared by Amicon Co.)
  • the concentrate was separated by column chromatography with Superose 12 (2.6 x 80 cm) pre-equilibrated with 50 mM phosphate buffer (pH 6.0) to obtain a single broad peak around 0.74 of the column volume. This was concentrated by using ultrafiltration (YM-10) to obtain the final product.
  • the concentration of the final product was determined by the color method using bovine serum albumin as a standard (prepared by Biorad Co.). The yields were 5 mg. The recovery indicated by biological activity was 23% of the first culture supernatants.
  • Example 6 To the pure IL4 (2 ⁇ g) obtained in Example 6, the same amount of loading buffer (250 mM tris hydrochloric acid buffer (pH 6.8), 4%(w/v) SDS, 18%(v/v) glycerol, 1.4M 2-mercaptomethanol, 0.01%(w/v) bromophenol blue) was admixed. The mixture was heated for three minutes at 100°C, and then separated by using SDS/PAGE [10 - 20%, 1 mm, slubgel, product of Dai-ichi-Kagaku-Yakuhin]. A single band was obserbed by Coomasie Blue staining at 18 kd. This band was reconfirmed by Silver-stain and the purity was measured as over 98% by a densitometric scan (CS-910, Shimazu) (see Figure 4).
  • loading buffer 250 mM tris hydrochloric acid buffer (pH 6.8), 4%(w/v) SDS, 18%(v/v)
  • the above N-terminal sequence is completely identical with that of human IL4 obtained by culturing COS-7 cells [see Proc. Natl. Acad. Sci., 83, 5394 (1986)].
  • the above result shows that the separation of a signal peptide between the 24th amino acid, glycine and the 25th amino acid, histidine, both deduced from cDNA sequence, was carried out completely.
  • the molecular weight of only the protein moiety deduced from the cDNA is 14962. Hence, the difference in molecular weight, i.e. about 3000 (about 17%) is considered to be the molecular weight of the sugar chain, which means the sugar content.
  • the analysis was carried out by using a TSK-gel sugar AXG column (prepared by Toyo Jozo Co.) in 0.5M borate buffer (pH 8.7). As a result, it was confirmed that the sample comprises mannose, fucose and galactose.
  • HPLC analysis was carried out by using a TSK-gel SCX column (prepared by Toyo Jozo Co.) in 0.16M borate buffer (pH 7.6). As a result, it was confirmed that the sample comprises glucosamine.
  • NANA N-acetylneuraminic acid

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Claims (21)

1. Humaninterleukin 4, gekennzeichnet durch
(a) ein Gesamtmolekulargewicht von 17 000 bis 19 000 und
(b) einen Zuckergehalt enstsprechend einem Molekulargewicht von 2 000 bis 4 000.
2. Humaninterleukin 4 nach Anspruch 1, wobei sein Zukkergehalt einem Molekulargewicht von 2 500 bis 3 500 entspricht.
3. Humaninterleukin 4, nach Anspruch 1 oder 2, weiterhin gekennzeichnet durch
(a) einen isoelektrischen Punkt von 8,5 bis 10,5.
4. Humaninterleukin 4 nach Anspruch 1 oder 2, wobei der isoelektrische Punkt 9,8 bis 10,1 beträgt.
5. Humaninterleukin 4 nach einem der Ansprüche 1 bis 4, weiterhin dadurch gekennzeichnet, daß
(d) sein Zuckeranteil mindestens Mannose, Fucose und Galactose als Neutralzucker umfaßt.
6. Humaninterleukin 4 nach einem der Ansprüche 1 bis 4, weiterhin dadurch gekennzeichnet, daß
(e) sein Zuckeranteil mindestens Glucosamin als Aminozucker umfaßt.
7. Humaninterleukin 4 nach einem der Ansprüche 1 bis 4, weiterhin dadurch gekennzeichnet, daß
(f) sein Zuckeranteil mindestens N-Acetylneuraminsäure als eine Sialsäure umfaßt.
8. Humaninterleukin 4 nach einem der Ansprüche 1 bis 4, weiterhin gekennzeichnet dadurch, daß
(d) sein Zuckeranteil mindestens Mannose, Fucose und Galactose als Neutralzucker,
(e) sein Zuckeranteil mindestens Glucosamin als Aminozucker und
(f) sein Zuckeranteil mindestens N-Acetylneuraminsäure als eine Sialsäure umfaßt.
9. Humaninterleukin 4 nach Anspruch 8, weiterhin gekennzeichnet durch
(g) die Anwesenheit von Mannose, Fucose, Galactose, Glucosamin bzw. N-Acetylneuraminsäure in Mengen von 3,0 bis 4,0, 1,0 bis 2,0, 2,5 bis 3,5, 4,5 bis 5,5 bzw. 1,5 bis 2,5 Mol(en) pro Gesamtmolmenge Zucker.
10. Humaninterleukin 4 nach Anspruch 1, hergestellt unter Verwendung eines Transformanten, der durch Transfektion von Eierstockzellen des Chinesischen Hamsters mit Dihydrofolatreduktasedefekt mit einem Expressionsvektor für Humaninterleukin 4 erhältlich ist, als Wirt, wobei der Expressionsvektor
(I) eine DNA-Sequenz mit Codierung für Humaninterleukin 4 an einer Stelle stromabwärts von einem SV40 Frühpromotor und
(11) eine DNA-Sequenz mit Codierung für Dihydrofolatreduktase an einer Stelle stromabwärts von einem in eukaryotischen Zellen operierenden Promotor umfaßt.
11. Humaninterleukin 4 nach Anspruch 10, wobei in dem Expressionsvektor für Humaninterleukin 4 mindestens die Humaninterleukin-4-Gennucleotidsequenz für Instabilität entfernt ist.
12. Humaninterleukin 4 nach Anspruch 10 oder 11, weiterhin gekennzeichnet durch
(c) einen isoelektrischen Punkt von 8,5 bis 10,5.
13. Humaninterleukin 4 nach einem der Ansprüche 10 bis 12, weiterhin dadurch gekennzeichnet, daß
(d) sein Zuckeranteil mindestens Mannose, Fucose und Galactose als Neutralzucker umfaßt.
14. Humaninterleukin 4 nach einem der Ansprüche 10 bis 12, weiterhin dadurch gekennzeichnet, daß
(e) sein Zuckeranteil mindestens Glucosamin als Aminozucker umfaßt.
15. Humaninterleukin 4 nach einem der Ansprüche 10 bis 12, weiterhin dadurch gekennzeichnet, daß
(f) sein Zuckeranteil mindestens N-Acetylneuraminsäure als eine Sialsäure umfaßt.
16. Humaninterleukin 4 nach einem der Ansprüche 10 bis 12, weiterhin dadurch gekennzeichnet, daß
(d) sein Zuckeranteil mindestens Mannose, Fucose und Galactose als Neutralzucker,
(e) sein Zuckeranteil mindestens Glucosamin als Aminozucker und
(f) sein Zuckeranteil mindestens N-Acetylneuraminsäure als eine Sialsäure umfaßt.
17. Humaninterleukin 4 nach Anspruch 16, weiterhin gekennzeichnet durch
(g) die Anwesenheit von Mannose, Fucose, Galactose, Glucosamin bzw. N-Acetylneuraminsäure in Mengen von 3,0 bis 4,0, 1,0 bis 2,0, 2,5 bis 3,5, 4,5 bis 5,5 bzw. 1,5 bis 2,5 Mol(en) pro Gesamtmolmenge Zucker.
18. Expressionsvektor für Humaninterleukin 4, gekennzeichnet durch
(a) ein Gesamtmolekulargewicht von 17 000 bis 19 000 und
(b) seinen Zuckergehalt enstsprechend einem Molekulargewicht von 2 000 bis 4 000, umfassend
(I) eine DNA-Sequenz mit Codierung für Humaninterleukin 4 an einer Stelle stromabwärts von einem SV40 Frühpromotor und
(11) eine DNA-Sequenz mit Codierung für Dihydrofolatreduktase an einer Stelle stromabwärts von einem in eukaryotischen Zellen operierenden Promotor.
19. Expressionsvektor nach Anspruch 18, wobei mindestens die Humaninterleukin-4-Gennucleotidsequenz für Instabilität entfernt ist.
20. Transformant, der durch Transfektion von Eierstockzellen des Chinesischen Hamsters mit Dihydrofolatreduktasedefekt mit einem Expressionsvektor für Humaninterleukin 4 erhältlich ist, gekennzeichnet durch
(a) ein Gesamtmolekulargewicht von 17 000 bis 19 000 und
(b) seinen Zuckergehalt enstsprechend einem Molekulargewicht von 2 000 bis 4 000, umfassend
(I) eine DNA-Sequenz mit Codierung für Humaninterleukin 4 an einer Stelle stromabwärts von einem SV40 Frühpromotor und
(11) eine DNA-Sequenz mit Codierung für Dihydrofolatreduktase an einer Stelle stromabwärts von einem in eukaryotischen Zellen operierenden Promotor.
21. Transformant nach Anspruch 20, wobei in dem Expressionsvektor für Humaninterleukin 4 mindestens die Humaninterleukin-4-Gennucleotidsequenz für Instabilität entfernt ist.
EP88112458A 1987-08-03 1988-08-01 Menschliches Interleukin-4, Expressionsvektoren dafür und diese enthaltende Transformanten Expired - Lifetime EP0302429B1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT88112458T ATE92110T1 (de) 1987-08-03 1988-08-01 Menschliches interleukin-4, expressionsvektoren dafuer und diese enthaltende transformanten.

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP192666/87 1987-08-03
JP19266687 1987-08-03
JP39482/88 1988-02-24
JP3948288 1988-02-24

Publications (2)

Publication Number Publication Date
EP0302429A1 EP0302429A1 (de) 1989-02-08
EP0302429B1 true EP0302429B1 (de) 1993-07-28

Family

ID=26378886

Family Applications (1)

Application Number Title Priority Date Filing Date
EP88112458A Expired - Lifetime EP0302429B1 (de) 1987-08-03 1988-08-01 Menschliches Interleukin-4, Expressionsvektoren dafür und diese enthaltende Transformanten

Country Status (4)

Country Link
EP (1) EP0302429B1 (de)
KR (1) KR890003804A (de)
DE (1) DE3882611T2 (de)
ES (1) ES2059453T3 (de)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7744899B2 (en) 1998-12-31 2010-06-29 The University Of Chicago Recombinant herpes simplex virus useful for treating neoplastic disease

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2218420B (en) * 1988-04-12 1992-07-15 British Bio Technology Synthetic gene encoding interleukin 4
ES2082201T3 (es) * 1990-03-21 1996-03-16 Schering Corp Uso de il-4 para mejorar la respuesta inmune a las pruebas de antigenos infecciosos.
US5206345A (en) * 1990-08-02 1993-04-27 Fred Hutchinson Cancer Research Center Il-4 and tnf induce mab 6g10-recognized expression on bone marrow stromal cells
US5827670A (en) * 1990-08-02 1998-10-27 Fred Hutchinson Cancer Research Center Methods of isolating and detecting bone marrow stromal cells with VCAM-1-specific antibodies
US7449186B1 (en) 1990-08-02 2008-11-11 Fred Hutchinson Cancer Research Center Methods of blocking the interaction between stromal cells and hemopoietic cells with anti-VCAM-1 antibodies
AU661979B2 (en) * 1990-09-06 1995-08-17 Schering Corporation Use of IL-4 to treat solid tumors
DE69209051T2 (de) * 1991-01-10 1996-08-08 Schering Corp Verwendung von il-4 zur verstärkung von wundheilung und besserung und zur heilung von infizierten wunden und wunden bei diabetischen säugetieren
US5494662A (en) * 1992-04-27 1996-02-27 Ono Pharmaceutical Co., Ltd. Stimulator for bone formation
US7361331B2 (en) 1996-10-18 2008-04-22 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Plant bioreactors

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PT83761B (pt) * 1985-11-19 1989-06-30 Schering Biotech Corp Metodo para a producao de interleuquina-4 de mamifero
ZA872781B (en) * 1986-05-19 1987-10-05 Immunology Ventures B-cell stimulating factor

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7744899B2 (en) 1998-12-31 2010-06-29 The University Of Chicago Recombinant herpes simplex virus useful for treating neoplastic disease

Also Published As

Publication number Publication date
KR890003804A (ko) 1989-04-18
DE3882611T2 (de) 1993-12-23
ES2059453T3 (es) 1994-11-16
EP0302429A1 (de) 1989-02-08
DE3882611D1 (de) 1993-09-02

Similar Documents

Publication Publication Date Title
EP0077670B1 (de) Humanes Immuninterferon
EP0261592B1 (de) Für menschliche "Colony-stimulating"-Faktoren kodierendes Gen
EP0285448B1 (de) Leukämie hemmender Faktor
Rajavashisth et al. Cloning and tissue-specific expression of mouse macrophage colony-stimulating factor mRNA.
KR0184235B1 (ko) 거핵구 형성 인자
JP2655591B2 (ja) 霊長動物造血成長因子の新規種族
US20070104680A1 (en) Antibodies to natural killer stimulatory factor
KR920003822B1 (ko) 인체 과립구 대식 세포 및 호산성 세포 성장 인자 활성을 나타내는 폴리펩티드
EP0302429B1 (de) Menschliches Interleukin-4, Expressionsvektoren dafür und diese enthaltende Transformanten
CA1282356C (en) INTERFERON .alpha. 76
EP0276846A2 (de) Derivate des koloniestimulierenden Faktors
CA1282354C (en) INTERFERON .alpha. 54
EP0427189A1 (de) Geänderte Formen des menschlichen Erythropoietins und DNA-Sequenzen, die Gene kodieren, die diese Proteine exprimieren können
CA1282355C (en) INTERFERON .alpha. 61
JPH082310B2 (ja) ヒト顆粒球哺乳類宿主細胞中におけるマクロファージコロニー刺激因子産生のための発現ベクター
JP4326470B2 (ja) 糖鎖化ヒト顆粒球コロニー刺激因子同種体
JPH10500850A (ja) インターロイキン−６ スプライス変異体
EP0328061A2 (de) Menschlicher Koloniestimulierungsfaktor
KR960008010B1 (ko) IL-1α 유도체 및 혈소판 감소증 치료 약제
EP0098864A1 (de) Interferon-alpha 74
EP0261625B1 (de) Menschlicher B-Zellen-Differenzierungsfaktor und Verfahren zu dessen Herstellung
US5650297A (en) DNA encoding human colony-stimulating factors
EP0308424A1 (de) Ein neues protein, neuroleukin
JP3689111B2 (ja) インターロイキン１５
JPS60126088A (ja) 新規ｄνａおよびその用途

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE ES FR GB GR IT LI LU NL SE

17P Request for examination filed

Effective date: 19890728

17Q First examination report despatched

Effective date: 19920320

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH DE ES FR GB GR IT LI LU NL SE

REF Corresponds to:

Ref document number: 92110

Country of ref document: AT

Date of ref document: 19930815

Kind code of ref document: T

RIN1 Information on inventor provided before grant (corrected)

Inventor name: KAWASAKI, AKIYOSHI,

Inventor name: HAMA, KAZUAKI,

Inventor name: HONJO, TASUKU,

ITF It: translation for a ep patent filed

Owner name: SOCIETA' ITALIANA BREVETTI S.P.A.

ITTA It: last paid annual fee
REF Corresponds to:

Ref document number: 3882611

Country of ref document: DE

Date of ref document: 19930902

REG Reference to a national code

Ref country code: GR

Ref legal event code: FG4A

Free format text: 3008794

ET Fr: translation filed
EPTA Lu: last paid annual fee
PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed
REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2059453

Country of ref document: ES

Kind code of ref document: T3

EAL Se: european patent in force in sweden

Ref document number: 88112458.0

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 19950721

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 19950809

Year of fee payment: 8

Ref country code: DE

Payment date: 19950809

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: AT

Payment date: 19950811

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 19950814

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 19950816

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 19950825

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: ES

Payment date: 19950830

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GR

Payment date: 19950831

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 19950911

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: LU

Payment date: 19951001

Year of fee payment: 8

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 19960801

Ref country code: GB

Effective date: 19960801

Ref country code: AT

Effective date: 19960801

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Effective date: 19960802

Ref country code: ES

Free format text: LAPSE BECAUSE OF THE APPLICANT RENOUNCES

Effective date: 19960802

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Effective date: 19960831

Ref country code: CH

Effective date: 19960831

Ref country code: BE

Effective date: 19960831

BERE Be: lapsed

Owner name: ONO PHARMACEUTICAL CO. LTD

Effective date: 19960831

Owner name: HONJO TASUKU

Effective date: 19960831

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: THE PATENT HAS BEEN ANNULLED BY A DECISION OF A NATIONAL AUTHORITY

Effective date: 19970228

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Effective date: 19970301

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 19960801

REG Reference to a national code

Ref country code: GR

Ref legal event code: MM2A

Free format text: 3008794

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Effective date: 19970430

NLV4 Nl: lapsed or anulled due to non-payment of the annual fee

Effective date: 19970301

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Effective date: 19970501

EUG Se: european patent has lapsed

Ref document number: 88112458.0

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 19991007

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20050801